Increased oxygen free radical and nitric oxide (NO) generation has been hypothesized to be a central mechanism of the injury which occurs on reperfusion of ischemic tissues. Questions and controversy remain, however, regarding the role of this radical generation in the processes of myocardial stunning and infarction and how best to intervene to prevent radical generation and subsequent injury. Within the present grant, EPR techniques were developed and applied to measure the process of oxygen radical and NO generation in the isolated heart. New instrumentation enabling in-vivo EPR spectroscopy and imaging of free radicals in whole beating hearts at L-band (1-2 GHz) was also developed. In this renewal application, we propose studies to determine the role of oxygen radical and NO generation in the process of myocardial stunning and infarction, extending these observations from isolated buffer perfused to blood perfused hearts, and then to the setting of in-vivo regional ischemia as occurs with acute occlusion of a coronary artery. Direct and spin trapping EPR studies at X-band, as well as in vivo EPR studies at L-band will be performed to measure, quantitate, and characterize oxygen radical and NO generation in isolated hearts and in- vivo animal models of myocardial stunning and infarction. The importance and inter-relationship of the cellular mechanisms of this radical generation will be determined in order to develop optimal therapeutic approaches to prevent radical generation and radical mediated injury. Radical generation will be correlated with alterations in contractile function, high energy phosphates, and the metabolic state of the heart. Cell death will be measured by enzyme release, and histology. In-vivo EPR spectroscopy and imaging will be applied to measure and spatially map myocardial oxygenation, radical metabolism, NO formation, and cell death during ischemia and reflow. These experiments will provide new insight into the fundamental nature and mechanisms of oxygen free radical and nitric oxide generation in the postischemic heart and their role in myocardial stunning and infarction.